Method and composition for treating a bone tissue defect

ABSTRACT

The invention is directed to a biodegradable implant precursor having a two-part structure made of an outer sac and a liquid content. The implant precursor is composed of a biodegradable, water-coagulable thermoplastic polymer and a water-miscible organic solvent. When administered to an implant site in an animal, the implant precursor will solidify in situ to a solid, microporous matrix by dissipation of the organic solvent to surrounding tissue fluids and coagulation of the polymer. The invention also includes methods of making the implant precursor, an apparatus for forming the precursor, and a kit containing the apparatus. Also provided are methods of using the implant precursor for treating a tissue defect in an animal, for example, for enhancing cell growth and tissue regeneration, wound and organ repair, nerve regeneration, soft and hard tissue regeneration, and the like, for delivery of biologically-active substances to tissue or organs, and other like therapies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a Continuation of application Ser. No.08/294,754, filed Aug. 23, 1994 U.S. Pat. No. 5,660,849, (incorporatedherein by reference) which is a continuation-in-part of U.S. patentapplication Ser. No. 07/783,512, filed Oct. 28, 1991 U.S. Pat. No.5,324,519, which is a continuation-in-part of U.S. patent applicationSer. No. 07/384,416, filed Jul. 24, 1989 (now U.S. Pat. No. 5,077,049).

BACKGROUND OF THE INVENTION

In the course of periodontal disease, infection of gingival tissue byplaque bacteria causes the ligaments attaching the gum and teeth torecede, decalcifies the bony structure holding the teeth roots to thebone, and forms periodontal pockets in the gingival tissue adjacent theteeth. Successful periodontal restoration is known to occur ifperiodontal ligament cells are allowed to colonize root surfacespreferentially over gingival epithelial cells, gingival fibroblasts orosteoblasts. Surgery alone, however, does not result in restoration oflost periodontium.

In an attempt to promote and achieve periodontal restoration, implanttechniques have been developed. For example, microporous membranes, suchas the Millipore® filter and GORE-TEX® membranes, have been developedfor use in periodontal tissue regeneration. Typically, the periodontalflap is cut, and the microporous membrane is surgically inserted tocover the surface of the tooth root and to physically occlude epithelialcells from apically migrating along the root surface.

These membranes have several drawbacks. Besides providing variableresults, a second surgical entry is needed to remove the membrane aftertissue regeneration has been achieved because the membranes are notbiodegradable. There is also a higher incidence of infection inconnection with their use.

To preclude surgical removal of an implant, membranes made ofbioabsorbable material, such as microfibrillar collagen, polylacticacid, and polygalactin (Vicryl®) mesh have been used. Fitting andpositioning these membranes to the implant site is cumbersome andtime-consuming, and the therapeutic effect of these membranes has beenunpredictable. In addition, the degradation time of membranes composedof collagen has been variable, and the risk of adverse immunologicalreaction to this foreign protein material in the body presents a majorconcern.

A liquid system containing a biodegradable polymer has been developedwherein the solution is injected into an implant site, and solidifies insitu to form a biodegradable implant having a solid microporous matrix.Advantageously, the implant does not require surgical removal. However,controlled delivery and containment of a liquid system within aparticular area within the implant site is difficult, and the liquid mayspread to areas other than the implant site.

Therefore, there is a need for an article which will facilitate thecontrolled placement in an implant site of a liquid polymer solution forforming an implant. A further need is to develop a precursor to a solidimplant which is neither all-liquid nor all-solid but will solidify insitu to form a solid microporous implant. There is also a need for aprecursor to a solid implant that can be applied to a tissue defect inan animal and shaped or molded in situ to conform to the defect. Yetanother need is to develop in vivo and ex vivo methods of making animplant precursor having such characteristics.

SUMMARY OF THE INVENTION

These and other goals are achieved by the present invention which isdirected to an implant precursor for implantation in an animal, such asa human or other mammal, which will eventually harden in situ to a solidimplant having a microporous matrix. The invention also provides amethod of making and using the implant precursor. An apparatus is alsoprovided for forming an implant precursor ex vivo, and a kit containingthe apparatus.

The implant precursor is a two-part structure composed of an outer sacwith a liquid content. The implant precursor is composed of abiocompatible, biodegradable and/or bioerodible, water-coagulablethermoplastic polymer or copolymer which is substantially insoluble inan aqueous media, and a pharmaceutically-acceptable, water-solubleorganic solvent. The two-part structure of the implant precursor isformed by contacting a portion of a water-coagulable polymer solutionwith water or other aqueous medium, whereupon the solvent dissipatesinto the aqueous medium. This causes the polymer on the surface of theportion of polymer solution adjacent the aqueous medium to coagulate toform an outer sac having a firm consistency ranging from gelatinous towaxen-like, while the solution inside the sac (i.e., sac contents)remains a liquid. The sac contents of the implant precursor may range inconsistency from watery to slightly viscous.

The implant precursor may be applied to an implant site in an animal,such as a void, a defect, surgical incision, and the like, in or on ahard or soft tissue. Once placed in the implant site, the implantprecursor eventually forms a solid microporous implant by thedissipation of the organic solvent into surrounding tissue fluids andthe further coagulation of the polymer. Preferably, the matrix of theresulting implant has a two-layered pore structure with a highly porousinner core portion and a comparatively less porous outer surface layeror skin. Pores are formed in the solid matrix of the implant bydissipation of the solvent out of the composition into surroundingtissue fluids. Optionally, the implant precursor may include a separatepore-forming agent that is capable of generating pores within thepolymer matrix of the solid implant, as for example, sucrose, sodiumchloride, a cellulose-based polymer, and the like.

The resulting solid implant is biodegradable, bioabsorbable, and/orbioerodible, and will be gradually absorbed into the surrounding tissuefluids, as for example, blood serum, lymph, cerebral spinal fluid (CSF),saliva, and the like, and become disintegrated through enzymatic,chemical or cellular hydrolytic action. Generally, the implant will beabsorbed over a period of up to about 2 years to about 3 years,preferably within about 1-9 months, preferably within about 60-180 days.The implant may be used, for example, for selective enhancement of cellgrowth and tissue regeneration, delivery of biologically-activesubstances to the animal, and the like.

The implant precursor may also include a biologically-active agent, orbioactive agent, as for example, an anti-inflammatory agent, anantiviral agent, an antibacterial or antifungal agent useful fortreating and preventing infections in the implant site, a growth factor,a hormone, and the like. The implant resulting from the in situcoagulation of the implant precursor, may then serve as a system fordelivering the biologically-active agent to the animal.

A release rate modification agent may also be included in the implantprecursor for controlling the rate of breakdown of the implant matrixand/or the rate of release of a bioactive agent in vivo from the implantmatrix. Examples of suitable substances for inclusion as a release ratemodification agent include dimethyl citrate, triethyl citrate,ethyl-heptanoate, glycerin, hexanediol, and the like.

The invention also includes a method of making the implant precursor.The implant precursor may be formed in vivo or ex vivo by (a) coatingthe surface of a suitable support substrate with an effective amount ofan aqueous medium to form a layer; (b) dispensing onto the aqueouslayer, an effective amount of a liquid polymer solution made of awater-coagulable, biodegradable thermoplastic polymer such aspolylactide, polycaprolactone, polyglycolide, or copolymer thereof, anda water-soluble, pharmaceutically-acceptable organic solvent such asN-methyl-2-pyrrolidone; (c) applying an effective amount of an aqueousmedium onto the surface of the polymer solution; and (d) allowing thepolymer adjacent the aqueous medium to coagulate to form the implantprecursor having an outer sac with a liquid content. Preferably, thethickness of the implant precursor is controlled, for example, bycompressing the coagulating polymer mass between two solid flat surfacessuch as a glass plate, porous plastic, and the like. The aqueous mediumis applied onto the surface of the support substrate and the surface ofthe polymer solution in a minor but effective amount to initiatecoagulation of the polymer to form the outer sac of the implantprecursor.

The implant precursor may be formed in vivo by dispensing the polymersolution onto a soft or hard tissue or other support substrate in thebody of an animal. The precursor may also be formed ex vivo bydispensing the polymer solution onto a support substrate made, forexample, from glass, a porous plastic, sintered stainless steel,porcelain, bone material, and other like materials.

In a variation of forming an implant precursor, an amount of theforegoing liquid polymer solution is applied to the surface of thesupport substrate to form a line which delineates a boundary around adefined area. The implant precursor may then be formed within theconfines of the boundary line area. When the foregoing boundary line isformed on a tissue defect in vivo, an implant precursor may be preparedoutside the body and applied to the defect within the confines of theboundary line.

Optionally, a support layer may be applied to the tissue surface toprovide an adhesive substrate for securing the implant precursor ontothe surface of the tissue defect. Useful substances for forming anadhesive support layer include, for example, the foregoing liquidpolymer solution, a water-soluble substance such as gelatin, and thelike. The support layer may be in the form of a bead, a film or coating,and the like, having a thickness as desired.

The invention also includes an apparatus for forming an implantprecursor ex vivo. The apparatus is preferably a two-part assemblycomprising support means for maintaining the polymer solution on asurface during formation of an implant precursor such as a porous plateor block, and means for compressing the polymer solution duringformation of the implant precursor. Preferably, the support means andcompressing means are connected together by hinging means positionedalong one edge of the support means and the compressing means, such thatthe compressing means may be pivoted and placed onto the polymersolution on the support means. The support means and/or compressingmeans are preferably made of a porous material, as for example, a porousplastic, sintered stainless steel, porcelain, and other like materialswhich are absorptive to water. An aqueous medium is applied as a layerover the surface of the support means, the polymer solution applied overthe aqueous layer, and a second aqueous layer is applied over thepolymer solution. Preferably, two or more spacers such as a washer, arearranged on the surface of the support means to form a defined areathereinbetween, and the implant precursor is formed on the area betweenthe spacers. The compressing means is then positioned over the supportmeans with the spacers and coagulating polymer solution sandwichedthereinbetween, preferably compressing the coagulating polymer mass. Thesupport means and compressing means of the apparatus are maintained in asandwich arrangement until the outer sac of the implant precursor isformed. The support means and compressing means are then separated andthe resulting implant precursor is removed from the apparatus, trimmedas desired, and placed into the implant site.

Also provided is a kit containing, in combination, the precursor-formingapparatus, one or more barrier means, an amount of the aforedescribedpolymer solution in one or more vials or other containers, and an amountof an aqueous medium preferably a phosphate buffered saline in one ormore vials or other like container. The kit may also include a tweezersor other like means for picking up the formed implant precursor; acalibrated tweezers or other like means for measuring the dimensions ofthe tissue defect and/or the implant precursor; a gridded template orother like means for measuring the dimensions of the implant precursor;a scalpel, razor or other like means for trimming the implant precursorto a desired size; and/or a cotton pad or other like means for blottingthe aqueous medium from the surface of the implant precursor.

The invention also includes a method for treating a tissue defect in ananimal. The implant precursor may be used, for example, for enhancingcell growth and tissue regeneration, wound and organ repair, nerveregeneration, soft and hard tissue regeneration, and the like. Accordingto the invention, the foregoing implant precursor is applied to thetissue defect and allowed to coagulate to an implant having a solidmicroporous matrix.

As used herein, the term "implant site" is meant to include a site, inor on which the implant precursor is formed or applied, as for example,a soft tissue such as muscle or fat, or a hard tissue such as bone.Examples of implant sites include a tissue defect such as a tissueregeneration site; a void space such as a periodontal pocket, surgicalincision or other formed pocket or cavity; a natural cavity such as theoral, vaginal, rectal or nasal cavities, the cul-de-sac of the eye, andthe like; and other sites into which the implant precursor may be placedand formed into a solid implant. The term "biodegradable" means that thepolymer and/or polymer matrix of the implant will degrade over time bythe action of enzymes, by hydrolytic action and/or by other similarmechanisms in the human body. By "bioerodible," it is meant that theimplant matrix will erode or degrade over time due, at least in part, tocontact with substances found in the surrounding tissue fluids, cellularaction, and the like. By "bioabsorbable," it is meant that the polymermatrix will be broken down and absorbed within the human body, forexample, by a cell, a tissue, and the like.

Since the implant precursor does not flow like a liquid, it provideseasy manipulation and placement of a liquid polymer system for formingan implant on a select area of a tissue defect without the uncontrolledflow of the polymer solution outside the area of the implant site. Thepresent implant precursor provides a system for forming an implant witha desired thickness, size, and shape. Unlike a solid implant, theimplant precursor is easy to manipulate and may be shaped and moldedwithin the defect site as it solidifies. Advantageously, the moldabilityof the implant precursor allows it to conform to irregularities,crevices, cracks, holes, and the like, in the tissue defect site. Inaddition, the surface of the implant precursor is tacky to the touch andtends to remain in place where it is applied to a tissue defect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a side view of an embodiment of a precursor-forming apparatusof the invention.

FIG. 2 is a plan view of an alternate embodiment of theprecursor-forming apparatus, showing the placement of a series ofspacers thereon.

FIG. 3 is a side view of the precursor-forming apparatus of FIG. 2,showing the placement of the aqueous layers and polymer solution layerin the area between the spacers.

FIG. 4 is a side view of the precursor-forming apparatus of FIG. 3,showing the apparatus in a closed position during formation of animplant precursor.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an implant precursor in the form of anouter sac with a liquid content for implantation in an animal. The outersac of the implant precursor has a firm consistency ranging fromgelatinous to moldable and waxen-like. The implant precursor is composedof a biodegradable, water-coagulable, thermoplastic polymer incombination with a water-soluble, non-toxic organic solvent.

Upon implantation in the body of an animal, the organic solvent of theprecursor implant dissipates into surrounding tissue fluids and thepolymer coagulates to form a solid, microporous implant. The resultingsolid implant has a variety of uses, as for example, a barrier systemfor enhancing cell growth and tissue regeneration, delivery ofbiologically-active agents such as drugs and medicaments, and the like.

Polymer Solution

To prepare the implant precursor, a liquid polymer solution isformulated which comprises a biodegradable, water-coagulable,thermoplastic polymer, such as a polylactide, polycaprolactone,polyglycolide, or copolymer thereof, in combination with awater-soluble, non-toxic, organic solvent, such as N-methylpyrrolidone,as disclosed in U.S. Pat. No. 4,938,763 to Dunn et al. (issued Jul. 3,1990), the disclosure of which is incorporated by reference herein. Thepolymer solution may optionally include a pore-forming agent.

The polymers or copolymers are substantially insoluble in water and bodyfluids, and biodegradable and/or bioerodible within the body of ananimal. The implant precursor and resulting solid implant arebiocompatible in that neither the polymer, the solvent nor the polymermatrix cause substantial tissue irritation or necrosis at the implantsite.

Thermoplastic polymers

Thermoplastic polymers useful in the liquid polymer solution for formingthe implant precursor include pharmaceutically-compatible polymers thatare biodegradable, bioabsorbable, and soften when exposed to heat butreturn to the original state when cooled. The thermoplastic polymers arecapable of substantially dissolving in a water-soluble carrier, orsolvent, to form a solution. The thermoplastic polymers are also capableof coagulating, or solidifying, to form an outer sac having a firmconsistency ranging from gelatinous to waxen-like, and of eventuallycoagulating to a solid microporous matrix upon the dissipation of thesolvent component from the polymer solution, and the contact of thepolymer with an aqueous medium.

Thermoplastic polymers that are suitable for use in the polymer solutiongenerally include any having the foregoing characteristics. Examples arepolylactides, polyglycolides, polycaprolactones, polyanhydrides,polyamides, polyurethanes, polyesteramides, polyorthoesters,polydioxanones, polyacetals, polyketals, polycarbonates,polyorthoesters, polyphosphazenes, polyhydroxybutyrates,polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates,poly(malic acid), poly(amino acids), poly(methyl vinyl ether),poly(maleic anhydride), chitin, chitosan, and copolymers, terpolymers,or combinations or mixtures therein. Polylactides, polycaprolactones,polyglycolides and copolymers thereof are highly preferred thermoplasticpolymers.

The thermoplastic polymer is combined with a suitable organic solvent toform a solution. The solubility or miscibility of a polymer in aparticular solvent will vary according to factors such as crystallinity,hydrophilicity, capacity for hydrogen-bonding, and molecular weight ofthe polymer. Consequently, the molecular weight and the concentration ofthe polymer in the solvent are adjusted to achieve desired solubility.Highly preferred thermoplastic polymers are those that have a low degreeof crystallization, a low degree of hydrogen-bonding, low solubility inwater, and high solubility in organic solvents.

Solvents

Suitable solvents for use in the thermoplastic polymer solution arethose which are biocompatible, pharmaceutically-acceptable, misciblewith the polymer ingredient and water, and capable of diffusing into anaqueous medium, as for example, tissue fluids surrounding the implantsite, such as blood serum, lymph, cerebral spinal fluid (CSF), saliva,and the like. Preferably, the solvent has a Hildebrand (HLB) solubilityratio of from about 9-13(cal/cm³)^(1/2). The degree of polarity of thesolvent should be effective to provide at least about 10% solubility inwater, and to dissolve the polymer component.

Solvents that are useful in the liquid polymer solution include, forexample, N-methyl-2-pyrrolidone, 2-pyrrolidone, C₂ to C₆ alkanols,propylene glycol, acetone, alkyl esters such as methyl acetate, ethylacetate, ethyl lactate, alkyl ketones such as methyl ethyl ketone,dialkylamides such as dimethylformamide, dimethyl sulfoxide, dimethylsulfone, tetrahydrofuran, cyclic alkyl amides such as caprolactam,decylmethylsulfoxide, oleic acid, propylene carbonate, aromatic amidessuch as N,N-diethyl-m-toluamide, 1-dodecylazacycloheptan-2-one, and thelike. Preferred solvents according to the invention includeN-methyl-2-pyrrolidone, 2-pyrrolidone, dimethyl sulfoxide, ethyllactate,and propylene carbonate.

A mixture of solvents providing varying degrees of solubility for thepolymer components may be used to increase the coagulation rate ofpolymers that exhibit a slow coagulation or setting rate. For example,the polymer may be combined with a coagulant-promoting solvent systemcomposed of a mixture of a good solvent (i.e., solvent providing a highdegree of solubility) and a poorer solvent (i.e., solvent providing alow degree of solubility) or a non-solvent (i.e., one in which thepolymer is insolvent) relative to the polymer component. It is preferredthat the solvent mixture contain an effective amount of a good solventand a poorer or non-solvent, in admixture such that the polymer willremain soluble while in solution but coagulate upon dissipation ordiffusion of the solvents into surrounding tissue fluids at the implantsite.

The concentration of polymer in the liquid polymer composition willgenerally accomplish rapid and effective dissipation of the solvent andcoagulation of the polymer. This concentration may range from about 0.01gram of polymer per ml of solvent to an about saturated concentration,preferably from about 0.1 gram per ml to an about saturatedconcentration.

Upon contact with an aqueous medium such as water, a body fluid such asblood serum, lymph, and the like, the solvent diffuses from the polymersolution into the aqueous medium. This causes the polymer at the surfaceof the polymer solution and adjacent the aqueous medium to coagulate toform a two-part structure comprising an outer sac with a liquid content.The liquid content of the implant precursor may range in consistencyfrom watery to viscous. The outer sac may range in consistency fromgelatinous to an impressionable, moldable and waxen-like. The resultingdevice, or implant precursor, may then be applied to an implant site.Upon implantation, the solvent from the implant precursor diffuses intothe surrounding tissue fluids to form an implant having a solid polymermatrix. Preferably, the implant precursor solidifies in situ to a solidmatrix within about 0.5-4 hours after implantation, preferably withinabout 1-3 hours, preferably within about 2 hours.

Pore-formation and pore forming agents

When placed into an implant site in an animal, the implant precursoreventually coagulates to a solid, microporous matrix structure.Preferably, the matrix is composed of a microporous inner core portionand an outer microporous skin. The pores of the inner core portion arepreferably substantially uniform and the skin of the solid implant isessentially non-porous compared to the porous nature of the core.Preferably, the outer skin portion of the implant has pores withdiameters significantly smaller in size than these pores in the innercore portion.

Pores may be formed within the matrix of the implant by several means.The dissipation, dispersement or diffusion of the solvent out of thesolidifying polymer matrix into the adjacent tissue fluids may generatepores, including pore channels, in the polymer matrix. The dissipationof the solvent from the coagulating mass creates pores within the solidimplant. The size of the pores of the solid implant are in the range ofabout 1-1000 microns, preferably the size of pores of the skin layer areabout 3-500 microns. The solid microporous implant has a porosity in therange of about 5-95%.

Optionally, a pore-forming agent may be included in the polymer solutionto generate additional pores in the polymer matrix. The pore-formingagent may be any pharmaceutically-acceptable, organic or inorganic,water-soluble substance that is substantially soluble in water and bodyfluids, and will dissipate from the coagulating polymer matrix and/orthe solid matrix of the implant into surrounding body fluids at theimplant site. The porous matrices formed through the inclusion of apore-forming agent have a pore structure in which the pores aresubstantially similar in size.

It is preferred that the pore-forming agent is soluble or dispersible inthe organic solvent to form a uniform mixture with the polymer, eitheras a dispersion or suspension, or as a solution. The pore-forming agentmay also be a water-immiscible substance that rapidly degrades to awater-soluble substance. Preferably, the pore-forming agent is combinedwith the thermoplastic polymer and solvent in admixture, before thematrix is formed. Suitable pore-forming agents that may be used in thepolymer composition include, for example, sugars such as sucrose anddextrose, salts such as sodium chloride and sodium carbonate, polymerssuch as hydroxylpropylcellulose, carboxymethylcellulose, polyethyleneglycol, and polyvinylpyrrolidone, and the like. Solid crystals that willprovide a defined pore size, such as salt or sugar, are preferred.

When the implant precursor is applied to an implant site, the solventand/or pore-forming agent dissipates into surrounding tissue fluids.This causes the formation of microporous channels within the coagulatingpolymer matrix. Optionally, the pore-forming agent may dissipate fromthe matrix into the surrounding tissue fluids at a rate slower than thatof the solvent, or be released from the matrix over time bybiodegradation or bioerosion of the matrix. Preferably, the pore-formingagent dissipates from the coagulating implant matrix within a short timefollowing implantation such that a matrix is formed with a porosity andpore structure effective to perform the particular purpose of theimplant, as for example, a barrier system for a tissue regenerationsite, a matrix for timed-release of a drug or medicament, and the like.

Porosity of the solid implant matrix may be varied by the concentrationof water-soluble or water-miscible ingredients, such as the solventand/or pore-forming agent, in the polymer composition. For example, ahigh concentration of water-soluble substances in the thermoplasticcomposition may produce a polymer matrix having a high degree ofporosity. The concentration of the pore-forming agent relative topolymer in the composition may be varied to achieve different degrees ofpore-formation, or porosity, in the matrix. Generally, the polymercomposition will include about 0.01-1 gram of pore-forming agent pergram polymer.

The size or diameter of the pores formed in the matrix of the solidimplant may be modified according to the size and/or distribution of thepore-forming agent within the polymer matrix. For example, pore-formingagents that are relatively insoluble in the polymer mixture may beselectively included in the polymer composition according to particlesize in order to generate pores having a diameter that corresponds tothe size of the pore-forming agent. Pore-forming agents that are solublein the polymer mixture may be used to vary the pore size and porosity ofthe implant matrix by the pattern of distribution and/or aggregation ofthe pore-forming agent within the polymer mixture and coagulating andsolid polymer matrix.

Where the implant is used to promote guided tissue regeneration, it ispreferred that the diameter of the pores in the matrix are effective todeter growth of epithelial cells and enhance growth of connective tissuecells into the polymer matrix of the implant. It is further preferredthat the size of the pores and porosity of the matrix of the implantfacilitate diffusion of nutrients and other growth-promoting substancessuch as growth factors, to cells which have grown into the matrix.Preferably, the degree of porosity of the matrix provides an implantthat is capable of substantially maintaining structural integrity forthe desired period of time without breakage or fracturing during use.

To provide an effective implant for bone cell regrowth and tissueregeneration, it is preferred that the diameter of the pores of theimplant is about 3-500 microns, more preferably about 3-200 microns,more preferably about 75-150 microns. It is further preferred that thematrix has a porosity of about 5-95%, preferably about 25-85%, in orderto provide optimum cell and tissue ingrowth into the matrix and optimumstructural integrity.

Pore diameter and distribution within the polymer matrix of the solidimplant may be measured, as for example, according to scanning electronmicroscopy methods by examination of cross-sections of the polymermatrix. Porosity of the polymer matrix may be measured according tosuitable methods known in the art, as for example, mercury intrusionporosimetry, specific gravity or density comparisons, calculation fromscanning electronic microscopy photographs, and the like. Additionally,porosity may be calculated according to the proportion or percent ofwater-soluble material included in the polymer composition. For example,a polymer composition which contains about 30% polymer and about 70%solvent and/or other water-soluble components will generate an implanthaving a polymer matrix of about 70% porosity.

Biologically-active Agent.

Optionally, the polymer solution may include a biologically-activeagent, either singly or in combination, such that the implant precursorand implant will provide a delivery system for the agent to adjacent ordistant tissues and organs in the animal. Biologically-active agentswhich may be used alone or in combination in the implant precursor andimplant include, for example, a medicament, drug, or other suitablebiologically-, physiologically-, or pharmaceutically-active substancewhich is capable of providing local or systemic biological,physiological or therapeutic effect in the body of an animal including amammal, and of being released from the solid implant matrix intoadjacent or surrounding tissue fluids.

The biologically-active agent may be soluble in the polymer solution toform a homogeneous mixture, or insoluble in the polymer solution to forma suspension or dispersion. Upon implantation, the biologically-activeagent preferably becomes incorporated into the implant matrix. As thematrix degrades over time, the biologically-active agent is releasedfrom the matrix into the adjacent tissue fluids, preferably at acontrolled rate. The release of the biologically-active agent from thematrix may be varied, for example, by the solubility of thebiologically-active agent in an aqueous medium, the distribution of theagent within the matrix, the size, shape, porosity, solubility andbiodegradability of the implant matrix, and the like.

The polymer solution, implant precursor and implant include thebiologically-active agent in an amount effective to provide the desiredlevel of biological, physiological, pharmacological and/or therapeuticeffect in the animal. There is generally no critical upper limit on theamount of the bioactive agent included in the polymer solution. The onlylimitation is a physical limitation for advantageous application, i.e.,the bioactive agent should not be present in such a high concentrationthat the solution or dispersion viscosity is too high for injection. Thelower limit of the amount of bioactive agent incorporated into thepolymer solution will depend on the activity of the bioactive materialand the period of time desired for treatment.

The biologically-active agent may stimulate a biological orphysiological activity with the animal. For example, the agent may actto enhance cell growth and tissue regeneration, function in birthcontrol, cause nerve stimulation or bone growth, and the like. Examplesof useful biologically-active agents include a substance, or metabolicprecursor thereof, which is capable of promoting growth and survival ofcells and tissues, or augmenting the functioning of cells, as forexample, a nerve growth promoting substance such as a ganglioside, anerve growth factor, and the like; a hard or soft tissue growthpromoting agent such as fibronectin (FN), human growth hormone (HGH),protein growth factor interleukin-1 (IL-1), and the like; a bone growthpromoting substance such as hydroxyapatite, tricalcium phosphate, andthe like; and a substance useful in preventing infection at the implantsite, as for example, an antiviral agent such as vidarabine oracyclovir, an antibacterial agent such as a penicillin or tetracycline,an antiparasitic agent such as quinacrine or chloroquine.

Suitable biologically-active agents for use in the invention alsoinclude anti-inflammatory agents such as hydrocortisone, prednisone andthe like; anti-bacterial agents such as penicillin, cephalosporins,bacitracin and the like; antiparasitic agents such as quinacrine,chloroquine and the like; antifungal agents such as nystatin,gentamicin, and the like; antiviral agents such as acyclovir, ribarivin,interferons and the like; antineoplastic agents such as methotrexate,5-fluorouracil, adriamycin, tumor-specific antibodies conjugated totoxins, tumor necrosis factor, and the like; analgesic agents such assalicylic acid, acetaminophen, ibuprofen, flurbiprofen, morphine and thelike; local anaesthetics such as lidocaine, bupivacaine, benzocaine andthe like; vaccines such as hepatitis, influenza, measles, rubella,tetanus, polio, rabies and the like; central nervous system agents suchas a tranquilizer, B-adrenergic blocking agent, dopamine and the like;growth factors such as colony stimulating factor, platelet-derivedgrowth factor, fibroblast growth factor, transforming growth factor B,human growth hormone, bone morphogenetic protein, insulin-like growthfactor and the like; hormones such as progesterone, follicle stimulatinghormone, insulin, somatotropins and the like; antihistamines such asdiphenhydramine, chlorphencramine and the like; cardiovascular agentssuch as digitalis, nitroglycerine, papaverine, streptokinase and thelike; anti-ulcer agents such as cimetidine hydrochloride, isopropamideiodide, and the like; bronchodilators such as metaproternal sulfate,aminophylline and the like; vasodilators such as theophylline, niacin,minoxidil, and the like; and other like substances. For other examplesof biologically-active agents that may be used in the present invention,see Applicants' corresponding U.S. patent application Ser. No.07/783,512, filed Oct. 28, 1991, U.S. Pat. No. 5,324,519 the disclosureof which is incorporated by reference herein.

Accordingly, the formed implant may function as a delivery system ofdrugs, medicaments and other biologically-active agents to tissuesadjacent to or distant from the implant site. The biologically-activeagent is preferably incorporated into the polymer matrix, andsubsequently released into surrounding tissue fluids and to thepertinent body tissue or organ.

Control of release of the bioactive agent

The rate of breakdown of the implant and/or release of a bioactive agentin vivo may be controlled by varying the type and molecular weight ofthe polymer(s), by including a release rate modification agent, and/orvarying the combination and concentrations of ingredients that comprisethe polymer solution.

The rate of release of a bioactive agent from the implant matrix may bemodified by varying the molecular weight of the polymer included in thepolymer solution. It has been found that for implant matrices formedthrough intermediacy of the foregoing liquid polymer solution, therelease rate of a bioactive agent follows a "U" shaped curve as themolecular weight of the polymer increases. That is, the rate of releaseof the bioactive agent will decrease, pass through a minimum, and thenagain increase as the molecular weight of a polymer is increased. As aresult, a polymer solution can be formulated with an optimum polymermolecular weight range for the release of a bioactive substance over aselected length of time. For example, to achieve a relatively quickrelease of a bioactive agent from the implant matrix, a polymermolecular weight on either side of the minimum for that particularpolymer would be used in the polymer solution. For release of abioactive agent over a relatively long period of time, a polymermolecular weight at or about the minimum for the particular polymerwould be preferred.

With the present polymer system, the typical minimum rate of release ofa bioactive agent from the solid implant matrix occurs at an inherentviscosity (I.V. in deciliters/gm) of about 0.2 but can vary depending onthe ingredients of the polymer solution. To achieve a sustained releaseof the bioactive agent from the implant matrix, it is preferred toadjust the molecular weight of the polymer to at least about 0.1inherent viscosity (I.V.) or about 2,000 molecular weight as determinedby gel permeation chromatography (comparison to polystyrene). Typically,acceptable sustained release rates are obtained if the molecular weightof the polymer is below about 0.8 I.V., or a molecular weight of about100,000. More preferably, the molecular weight is adjusted to be withina range of about 0.1-0.5 I.V., for effective sustained release. For apoly(DL-lactide) or a lactide-co-glycolide system, the desired molecularweight range is about 0.1-0.5 I.V. If a molecular weight of a specificpolymer is chosen from these parameters and the release of the bioactivesubstance is too slow or too fast, the rate can be varied simply bydetermining a few experimental points along the U curve for that polymerand adjusting the molecular weight accordingly.

The molecular weight of a polymer can be varied by any of a variety ofmethods known in the art. The choice of method is typically determinedby the type of polymer solution being formulated. For example, if athermoplastic polymer is used that is biodegradable by hydrolysis, themolecular weight can be varied by controlled hydrolysis, such as in asteam autoclave. Typically, the degree of polymerization can becontrolled, for example, by varying the number and type of reactivegroups and the reaction times.

For other examples and further discussion of controlling the rate ofrelease of a bioactive agent from the implant matrix by varying thepolymer composition of the polymer solution, see Applicants'corresponding U.S. patent application Ser. No. 07/776,816, filed Oct.15, 1991, now abandoned the disclosure of which is incorporated byreference herein.

Release Rate Modification Agents

The polymer solution may include a release rate modification agent toprovide controlled, sustained release of a bioactive agent from thesolid implant matrix. Although not intended to be a limitation to thepresent disclosure, it is believed the release rate modification agentalters the release rate of a bioactive agent from the implant matrix bychanging the hydrophobicity of the polymer implant.

The use of a release rate modification agent may either decrease orincrease the release of the bioactive agent in the range of multipleorders of magnitude (e.g., 1 to 10 to 100), preferably up to a ten-foldchange, as compared to the release of a bioactive agent from a solidmatrix without the release rate modification agent. For example,naltrexone and doxycycline are substantially completely released from apolymer matrix comprised of poly(DL-lactide) within about 2-3 days exvivo. With the addition of a release rate modification agent such asethyl heptanoate which is hydrophobic to the polymer solution, andformation of the implant matrix through interaction of the polymersolution and an aqueous medium, the release rate of naltrexone ordoxycycline can be slowed to produce substantially complete release ofthe drug within about seven days. With the inclusion of a greater amountof a release rate modification agent into the polymer solution, the timeperiod of the release can be increased to about fourteen days. Otherrelease rate modification agents which are hydrophilic such aspolyethylene glycol may increase the release of the bioactive agent. Byan appropriate choice of the polymer molecular weight in combinationwith an effective amount of the release rate modification agent, therelease rate and extent of release of a bioactive agent from the implantmatrix may be varied, for example, from relatively fast to relativelyslow.

Useful release rate modification agents include, for example, organicsubstances which are water-soluble, water-miscible, or water insoluble(i.e., water immiscible), with water-insoluble substances preferred.

The release rate modification agent is preferably an organic compoundwhich will substitute as the complementary molecule for secondaryvalence bonding between polymer molecules, and increases the flexibilityand ability of the polymer molecules to slide past each other. Such anorganic compound preferably includes a hydrophobic and a hydrophilicregion so as to effect secondary valence bonding. It is preferred that arelease rate modification agent is compatible with the combination ofpolymers and solvent used to formulate polymer solution. It is furtherpreferred that the release rate modification agent is apharmaceutically-acceptable substance.

Useful release rate modification agents include, for example, fattyacids, triglycerides, other like hydrophobic compounds, organicsolvents, plasticizing compounds and hydrophilic compounds. Suitablerelease rate modification agents include, for example, esters of mono-,di-, and tricarboxylic acids, such as 2-ethoxyethyl acetate, methylacetate, ethyl acetate, diethyl phthalate, dimethyl phthalate, dibutylphthalate, dimethyl adipate, dimethyl succinate, dimethyl oxalate,dimethyl citrate, triethyl citrate, acetyl tributyl citrate, acetyltriethyl citrate, glycerol triacetate, di(n-butyl) sebecate, and thelike; polyhydroxy alcohols, such as propylene glycol, polyethyleneglycol, glycerin, sorbitol, and the like; fatty acids; triesters ofglycerol, such as triglycerides, epoxidized soybean oil, and otherepoxidized vegetable oils; sterols, such as cholesterol; alcohols, suchas C₆ -C₁₂ alkanols, 2-ethoxyethanol, and the like. The release ratemodification agent may be used singly or in combination with other suchagents. Suitable combinations of release rate modification agentsinclude, for example, glycerin/propylene glycol, sorbitol/glycerine,ethylene oxide/propylene oxide, butylene glycol/adipic acid, and thelike. Preferred release rate modification agents include dimethylcitrate, triethyl citrate, ethyl heptanoate, glycerin, and hexanediol.

The amount of the release rate modification agent included in thepolymer solution will vary according to the desired rate of release ofthe bioactive agent from the implant matrix. Preferably, the polymersolution contains about 0.5-15%, preferably about 5-10%, of a releaserate modification agent.

For other examples and further discussion of release rate modificationagents, or rate modifying agents, for use in the present invention, seeApplicants' corresponding U.S. patent application Ser. No. 07/776,816,filed Oct. 15, 1991 now abandoned, the disclosure of which isincorporated by reference herein.

Other factors for release rate modification

The release rate of the bioactive agent from the implant matrix may alsobe adjusted by varying the concentration of the polymer in the polymersolution. For example, the more dilute the polymer concentration, themore readily the bioactive agent will be released from the implantmatrix. For example, in a system containing about 5% flurbiprofen and apolymer concentration of about 55% poly(DL-lactide), a cumulativerelease of about 11.4% at day 1 and about 23% at day 7 may be provided.With a polymer concentration of about 45%, the cumulative percentrelease is about 23% at day 1 and about 40% at day 7.

This effect can be used in combination with other means to moreeffectively control the release of the bioactive agent from the implantmatrix as desired. For example, by adjusting the concentration of thepolymer and/or the bioactive agent, together with control of themolecular weight and the amount of the release rate modification agent,a wide range of release rates can be achieved.

The release rate of a bioactive agent from the implant matrix may alsobe varied by the addition of additives such as a pore forming agent, asdiscussed herein.

Formation of the Implant Precursor

A number of methods may be used to form the implant precursor. Ingeneral, the implant precursor is formed by dispensing a portion of theliquid polymer solution onto the surface of a support substrate. Anaqueous medium is then placed in contact with the polymer solution.Solvent then diffuses out of the polymer solution and the aqueous mediumdiffuses into the solution. This causes coagulation of the polymeradjacent to the aqueous medium to form the outer sac of the implantprecursor.

Suitable support substrates include, for example, hard or soft tissue ofthe animal or an ex vivo material, as for example, glass, stainlesssteel, porcelain, solid plastic or porous plastic. These ex vivomaterials may optionally have either an attached layer of a differentmaterial, such as a nylon filter, or a coating or a surface treatment oran additive that allows the support to absorb or wick an aqueous medium.Aqueous media can be blood, saliva or other body fluids when thesubstrate is in the animal. Aqueous media which can be used either invivo or ex vivo included water and saline solutions. Other aqueous mediacan be used if they cause coagulation of the polymer solution and areclinically acceptable.

The aqueous medium can be present at the surface of the supportsubstrate or inside the support substrate prior to the dispensing of thepolymer solution or the aqueous medium can be applied on top and aroundthe polymer solution after it is in place. In this last case coagulationof the bottom surface of the polymer solution requires the aqueousmedium to travel underneath the polymer solution.

The amount of aqueous medium used and the time that the polymer solutionand aqueous medium are held in contact depends on the composition of thepolymer solution and the aqueous medium, the nature of the supportsubstrate, the geometry of the apparatus, the amount and dimensions ofthe polymer solution and the consistency desired for the implantprecursor. For a given procedure and set of materials the consistency ofthe implant precursor can be varied from gelatinous to formable andimpression-retaining to fairly rigid by increasing the time the polymersolution and aqueous medium are in contact.

After the implant precursor has been formed, the aqueous medium may beremoved by tipping the support and/or the implant precursor to allow theaqueous layer to run off or by blotting the aqueous layer with anabsorbent material such as a cotton swab, gauze pad or a sponge. Theimplant precursor may optionally then be trimmed to the desired size andshape and then placed in the implant site. It is trimmed and implantedinto the animal within about 1 to 60 minutes, preferably 1 to 10minutes, of the conclusion of the coagulation process. If not implantedor placed back into contact with an aqueous medium, the implantprecursor will soften and eventually revert to an all liquid phase. Thisprocess is caused by an interaction between the outer sac layer and theliquid contents. The solvent and aqueous medium redistribute in theimplant precursor which destroys the sac formed in the coagulationprocess and results in one continuous liquid phase.

The dimensions of the implant precursor can be controlled by a number ofmethods. It is preferred that the thickness of the implant precursor isabout 300-1500 μm, preferably about 600-1200 μm. The length and widthdesired depend on the dimensions of the implant site in the animal. Inthe preferred methods the thickness is controlled during the coagulationprocess and the length and width are controlled in a subsequent trimmingstep. The polymer solution is dispensed onto a flat support substrateand a second flat piece of support substrate is placed on top of thepolymer solution and forced down causing the polymer solution to thinout and spread until the desired gap between the support substrates isobtained. This gap may be defined by spacers which hold the supportsubstrate pieces apart or other means. The aqueous medium may be presentduring this process or applied after this process. The coagulation ofthe polymer solution in this defined space results in a sheet of implantprecursor material with a center section of substantially uniformthickness with thinner portions at the edges. The implant precursor isthen cut out of the center section of the sheet using a razor blade,surgical prep blade, scalpel or other means. This trimming step allowscontrol of the implant precursor length, width and shape.

Alternate methods of controlling the dimensions of the implant precursorinclude dispensing the polymer solution onto a support substrate onwhich the desired area (i.e., width, length) have been defined by sometype of barrier. They can be then controlled as previously described orby drawing a flat article such as a spatula across the surface of thecoagulating polymer mass or like means. The polymer solution may also bedispensed into a recessed area or void as, for example, in a pre-castdie or mold or template, or other like device, which has the dimensions(i.e., width, length, depth or thickness) of the implant precursor.Additional amounts of the polymer solution may be applied to thesurface(s) or edges of the coagulating polymer mass to adjust thedimensions.

Various devices may be used to form the implant precursor. One suchdevice, which may be used ex vivo or in vivo, is a "tweezer wiper". A"tweezer wiper" is constructed by attaching a plate with a hole or awire loop to one blade of the tweezer at a right angle to the tweezerblades such that the second blade sweeps across the surface of the plateor wire loop when the tweezer blades are spread apart. The plate or wireloop is placed on the tissue or ex vivo support substrate os that thehole in the pate or the inside of the wire loop defines the area for theimplant precursor. The polymer solution is then dispensed into this areaand leveled off to control the thickness by passing the second blade ofthe tweezer over the polymer solution. An aqueous medium is then appliedto cause coagulation. Alternatively, the aqueous medium is applied priorto the leveling procedure. Once the implant precursor is sufficientlycoagulated the "tweezer wiper" is separated from the substrate. Theresulting implant precursor can then be left in place in the in vivocase or otherwise used according to the method of the invention.

In another embodiment of the invention, an implant precursor may beformed in vivo or ex vivo by forming a boundary line on the surface ofthe support substrate to contain the polymer solution within a confinedarea. To form the boundary line on a substrate, an amount of water orother aqueous medium is applied as a coating on the surface of thesupport substrate, the polymer solution is dispensed as a line over thewater layer to define a confined area, and an amount of water is thenapplied to the surface of the polymer solution resulting in surfacecoagulation of the polymer solution. The resulting boundary line is atwo-part, tube-like structure made of an outer sac with a liquid center.An implant precursor may then be formed within the confines of theboundary line by dispensing an amount of the polymer solution onto anaqueous layer coated on the support substrate within the boundary linearea, and applying an aqueous medium to the polymer layer to form thetwo-part structure of the implant precursor. Where the boundary line isformed in vivo on the surface of a tissue defect, an implant precursorformed ex vivo may also be applied to the defect within the area definedby the boundary line. It is preferred that the implant precursor andassociated boundary line are manually worked together as the polymerfurther coagulates to form the solid implant matrix such that thecoagulating mass will conform to the contours of the tissue defect andimplant site. Preferably, when treating a periodontal bone tissue defectby this method, the boundary line is applied to the root and ligamenttissue of the bone defect site.

Implant Precursor-forming Apparatus

According to the invention, a preferred method for making an implantprecursor ex vivo is by the use of an apparatus, as shown generally inFIG. 1. It is understood, however, that a variety of shapes, sizes andarrangements of the implant precursor-forming apparatus can beaccommodated according to the invention.

FIG. 1 is a schematic drawing of the preferred apparatus design, shownclosed as it would be during the coagulation process. The apparatusconsists of a case which consists of upper and lower sections (1 and 2)held together by a hinge (3) on one end and a latching mechanism (4 and5) on the other end. Each section contains a sheet of porous hydrophilicplastic (6 and 7). When the case is closed the two sheets of poroushydrophilic plastic (6 and 7) are held apart by the spacers (8 and 9) asshown in FIG. 1. This apparatus is used by opening the case and fillingthe pores in the porous hydrophilic plastic sheets (6 and 7) with anaqueous medium. The polymer solution is then dispensed onto the poroushydrophilic plastic sheet in the lower half of the case (7) and the caseis closed as shown in FIG. 1. The spacers (8 and 9) define the gap inwhich the polymer solution is held during the coagulation process andtherefore control the thickness of the implant precursor. Once thedesired coagulation has elapsed the case is opened and the implantprecursor is trimmed and then implanted.

FIG. 2 details an alternate embodiment of the general apparatus design.The components are labeled with the same numbers as in FIG. 1. Thisembodiment contains one section (10) which is not present in FIG. 1. Itis a trimming grid which is a portion of the case bottom (2). The caseconsists of an upper and lower section (1 and 2) joined with a hinge (3)formed by snapping the two case sections together. The case is composedof a gamma resistant polypropylene. Alternate case materials which canwithstand contact with the polymer solution and sterilization by gammairradiation and are fairly rigid could be used. The latch mechanism iscomposed of portions (4 and 5) of the two case sections (1 and 2) whichreadily snap together and apart to allow opening and closing of the caseand hold the case tightly closed during the coagulation process. Thehydrophilic porous plastic sheets (6 and 7) are flat, rigid sheets withthe hydrophilicity and porosity needed to allow an aqueous medium tofill the pores of the sheet and then allow exchange of the aqueousmedium and the solvent between the polymer solution and aqueous mediumand the solvent between the polymer solution and aqueous medium in thepores during the coagulation process. The porosity of the sheet is onefactor which controls the rate of coagulation. The porous plastic may beof an intrinsically hydrophilic polymer or a blend of a hydrophobicpolymer blended with or treated with a surfactant or other agent whichincreases hydrophilicity. THe material used in the preferred embodimentis a polyethylene blended with a surfactant. The spacers (8 and 9) arerectangles of gamma resistant polypropylene.

The trimming grid (10) is a flat portion of the lower case section (2).After the coagulation process the implant precursor is placed on thetrimming grid where it is trimmed to the desire shape, length and widthusing a surgical prep blade, razor blade or other like means. Thetrimming grid has a pattern of 1 mm squares which aid in trimming to thedesired dimensions. This pattern may be present as part of the caseitself or printed onto the case or printed on a label which is thenaffixed to the case. In the preferred embodiment the pattern is printedon a clear label which is affixed to the underside of the case bottom(2). The pattern is visible through the clear to slightly hazy casebottom (2) and the clear label material. Having the label or printing onthe underside of the case eliminates the possibility of physical orchemical interaction of the implant precursor and the label or printing.

The dimensions of the apparatus are dependent on the desired dimensionsof the implant precursor. For production of an implant precursor of anapproximate thickness of 675 μm with a length and width of approximately20 mm or less the following approximate dimensions are appropriate. Thespacers (8 and 9) are 675 μm thick, 0.5 cm wide and 2.5 cm long. Theporous plastic sheets are 4.5 cm long and 3.0 cm wide with a thicknessof 0.3 cm. The trimming grid pattern (10) is 3.5 cm by 3.5 cm. The casesections (1 and 2) are approximately 7.5 cm by 5 cm with cavities forthe porous plastic sheets (6 and 7) 30 cm deep. For proper thicknesscontrol the case must be designed to close such that the spacers (8 and9) are tightly held between two porous plastic sheets (6 and 7) so thatthe coagulation occurs in a gap which corresponds to the thickness ofthe spacers.

Adhesive Layer

To enhance adhesion of the implant precursor in the implant site, anadhesive layer may be applied to the surface of the tissue and theformed implant precursor is then placed over the support layer. Theadhesive layer preferably helps to maintain the position of the implantprecursor as it coagulates to a solid matrix in the implant site. Theadhesive layer comprises a bioabsorbable, biodegradable and/orbioerodible substance capable of adhering to both the surface of thetissue defect and to the surface of the implant precursor. An adhesivelayer may be formed, for example, by applying a minor but effectiveamount of the foregoing liquid polymer solution in the form of a bead oras a coating on the surface of the tissue defect.

Support Layer

To maintain the structure and form of the implant precursor or to formthe implant precursor directly in vivo, a support layer may be appliedto the surface of the tissue and the polymer solution or formed implantprecursor is then placed over the support layer. Materials suitable foruse in forming a support layer include, for example, a natural bodymaterial such as a clot of blood or other body fluid, a water-solublesubstance such as gelatin or water-soluble polymer, as for example,polyvinyl pyrrolidone, and other like materials.

A support layer of clotted blood may be formed, for example, bypuncturing the tissue with a needle to generate a minor but effectiveflow of blood which is then allowed to clot. A formed implant precursor,or the liquid polymer solution itself may be applied to the surface ofthe support layer in the implant site.

In another embodiment, granules or small pieces of a biodegradableporous material such as polylactic acid, oxidized cellulose or gelatinand the like, may be used to fill in a tissue defect or void, and then aformed implant precursor may be applied to the granular supportmaterial, or the polymer solution may be dispensed over the supportlayer to form the implant precursor.

Another useful support layer is a solid matrix having a porous,foam-like structure. Such a matrix may be provided, for example, bymixing air into the foregoing polymer solution to provide a foam-likeconsistency, and allowing the mixture to coagulate to a matrix havingrelatively large pores and/or cavities. Air bubbles may be incorporatedinto the polymer solution, for example, by vigorous stirring the polymersolution, by blowing air into the solution using a syringe, and otherlike means. It is preferred that an aqueous medium is applied to thesurface of the foamed mixture to cause the polymer to coagulate to forma matrix having large cavities.

Large pores may also be provided in a solid support matrix by combiningthe polymer solution with a gas-forming agent, as for example, a mixtureof citric acid and sodium carbonate or bicarbonate. When contacted withan aqueous medium, the gas-forming agent reacts to form gas bubbles suchas carbon dioxide within the coagulating polymer matrix.

Where a void space is desirable between the tissue defect and the solidimplant, the support layer is preferably formed of a water-soluble,and/or a highly resorbable material. For example, the support layer maycomprise a water-soluble substance that will dissolve within a few days,as for example an oxidized cellulose or gelatin material such asSurgicel™ or Gelfoam™, commercially available from Johnson & JohnsonCompany and the Upjohn Company; a water-soluble polymer such aspolyvinyl pyrrolidone, polyethylene glycol, and hydroxypropyl cellulose,and the like; and other like substances. Preferably, the water-solublesupport layer will dissolve within about 1-14 days, preferably about 2-4days, after implantation of the implant precursor.

In cases where it is desired to promote tissue ingrowth into a substratein the implant site, it is preferred that the support layer comprises aporous material which has a relatively longer rate of degradation.Suitable materials include, for example, a polylactic acid materialtypically applied to molar extraction sites to inhibit dry sockets, asfor example, Drilac™ which is commercially available from THMBiomedical, Inc. and a hydroxyapatite material such as Interpore 200which is commercially available from Interpore International.Advantageously, a support layer made of a porous material such aspolylactic acid or hydroxyapatite, allows the blood to infiltrate andclot within the matrix which provides a source of nutrients to promotetissue ingrowth. It is noted that tissue ingrowth into the supportmatrix will eventually break down the support layer.

Kit for forming an implant precursor

The invention also includes a kit for forming an implant precursor exvivo. The kit includes, in combination, (i) a precursor-formingapparatus, as described hereinabove, which is preferably a two-partapparatus hinged along one side; (ii) one or more spacer means formaintaining a gap or space between the two halves of the apparatus, forexample, a washer, rod, block, and the like; (iii) one or more vials orother like means containing the aforedescribed polymer solution; and(iv) one or more vials or other like means containing a source ofaqueous medium such as water, phosphate buffered saline, and the like.The kit may further include a tweezers or other like means for liftingand holding the formed implant precursor; a device for measuring thedimensions of the tissue defect and/or the implant precursor, as forexample, a calibrated tweezers and the like; a gridded template andother like means for measuring the dimensions of the implant precursor;a scalpel, razor blade or other like means for trimming and sizing theimplant precursor; and/or a cotton pad or like other means for removingthe aqueous medium from the surface of the implant precursor.

Use of the Implant Precursor

The implant precursor may be used for treating a variety of tissuedefects. The implant precursor may be applied to an implant site in ananimal, such as a void, a defect, surgical incision, and the like, in ahard or soft tissue, by known surgical techniques.

Preferably, once placed in the implant site, the implant precursor willbe substantially coagulated to a solid but moldable matrix, within about0.5-4 hours, more preferably about 0.75-3 hours, even more preferablyabout 1-2 hours.

For example, the implant precursor may be used in a method for treatinga bone tissue defect such as an arm or leg bone fracture, a toothdefect, and the like. Preferably, the bone tissue is surgicallyseparated from the adjacent soft tissue to expose the defect, and theimplant precursor is placed into the bone defect, whereupon the implantprecursor hardens in situ to a solid implant.

In a preferred use according to the invention, the implant precursor maybe used as a barrier system for guided tissue regeneration. The implantprecursor may be formed outside the body of the animal and thenadministered to an implant site such as a tissue with a void such as aperiodontal pocket, a soft-tissue defect, a surgical incision, a bonedefect and the like. Once administered to the tissue regeneration site,the implant precursor will solidify to form a solid, microporous matrixthat provides a surface over which the cell may grow. To enhanceregeneration of a hard tissue such as bone tissue, it is preferred thatthe solid implant matrix provides support for new cell growth that willreplace the matrix as it becomes gradually absorbed or eroded by bodyfluids.

One example of using the implant precursor as a barrier system is in thetreatment of a periodontal disease. For such treatment, the gingivaltissue overlying the root of the tooth is surgically incised from thetooth root and bone to form a gingival tissue envelope or pocket, and animplant precursor is placed into the pocket and against the bone. Afterplacement, the tissue is sutured to close the pocket, and the implantprecursor is allowed to harden to a solid, microporous implant.

The implant precursor may be manipulated in the implant site to conformit to the contours of the tissue defect. For example, in a periodontaldefect, the gingival tissue flap may be urged over the solidifyingimplant matrix placed against the exposed root and bone, and pressureapplied to the surface of the overlying tissue onto the solidifyingmatrix. The solidifying matrix is malleable and such manipulation shapesthe implant on one side to conform to the tissue defect and on the otherside to the contours of the overlying tissue. The tissue may beretracted to assess the profile (i.e., shape) of the implant matrix and,optionally, additional amounts of the polymer solution may be added tobuild up the matrix and fill in irregularities as needed. In cases wherethe implant precursor is too large, a portion of the congealing matrixmay be cropped along the edges of the overlying tissue, as for example,just above the gum line of a gingival tissue pocket. The tissue may thenbe secured in place over the implant matrix, as for example, by suturingthe tissue at either end of the pocket to hold the tissue and implant inplace.

To aid in the adhesion of the implant precursor to the surface of thetissue defect, a bead or coating of the foregoing polymer solution maybe applied over the defect to provide a tacky surface. The implantprecursor or liquid polymer solution may then be applied to the surfaceof bead or coating.

The implant precursor may be used for attaching a skin graft tounderlying tissue of a wound; and such use of the implant precursorhelps prevent seroma or hematoma formation, and speed the healingprocess. Preferably, the implant precursor includes a topical antibioticagent.

The implant precursor may also be used to enhance closure of a surgicalincision as, for example, an incision through the sternum for open heartsurgery, by stabilizing the sternum and promoting healing. In such use,the implant precursor is applied to the two sides of the sternum priorto closure of the sternum with metal wires and/or sutures. Preferably,the implant precursor includes a growth factor and/or an antibioticagent.

Advantageously, the implant precursor provides a means of adhering animplant article to a tissue generally covered with a mucous layer, asfor example, a gingival tissue. Also, the implant precursor provides forthe application of a liquid polymer solution in an implant site withoutthe uncontrolled flow of liquid into areas other than those identifiedfor treatment. For example, in the treatment of a periodontal defect,use of the present implant precursor will advantageously avoid theaccumulation of a polymer solution into spaces between tooth roots andthe periodontal region where the ligament cells are located. The presentprecursor implant also facilitates a better match of a barrier implantin a tissue defect site than other devices known and used in the art.

The microporous polymer matrix of the implant is capable ofbiodegradation, bioerosion and/or bioabsorption within the implant siteof the animal. The particular polymer and the molecular weight of thepolymer may be varied according to the desired duration or time intervalfor maintaining the solid polymer matrix within the implant site, as forexample, a few days or weeks to several years. When the implant is usedto enhance cell growth and tissue regeneration, it is preferred that thepolymer matrix will disintegrate at a rate effective to allowdisplacement of the matrix by cell growth from the adjacent cells ortissue.

Formulation of the liquid polymer solution for preparing the implantprecursor, and administration of the implant precursor and polymersolution in vivo will ultimately be according to the judgment andprotocol of the patient's attending health care professional such as aphysician, or if appropriate, a dentist. Choice of the particularformulation of ingredients will be made by the attending health careprofessional. Without a bioactive agent, the solid implant resultingfrom the implant precursor can function as a structure for promotion ofcell growth and tissue repair. With a bioactive agent, the implant willnot only function in such capacity but will also convey the propertiesof the bioactive agent.

The amounts and concentrations of ingredients in implant precursoradministered to the patient will generally be effective to accomplishthe task intended. If that task is to fill a void space, an implantprecursor of an appropriate size and an effective amount of ingredientswill be administered to accomplish this task. For administration of abioactive agent, the amounts and release rates will followrecommendations of the manufacturer of the bioactive agent. Generally,the concentration of a bioactive agent in the liquid polymer solutionwill be about 0.01-400 mg per gram of polymer solution.

The invention will be described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

EXAMPLE 1 Ex vivo Formation of an Implant Precursor with a PorousPolyethylene Substrate

A polymer mixture comprising about 37% poly(DL-lactide)(DL-PLA) andabout 63% N-methyl-2-pyrrolidone (NMP) was prepared. The DL-PLA had amolecular weight of about 65,000 daltons (inherent viscosity inchloroform of about 0.50 dL/g). Polypropylene containers were filledwith this polymer mixture such that each contained about 0.8 g of thepolymer mixture. These filled containers were then sterilized byexposure to gamma radiation at a level of 25-35 kGy, which result in afinal molecular weight of the DL-PLA of about 38,000 daltons (inherentviscosity in chloroform of about 0.34 dL/g).

The apparatus diagrammed in FIG. 2 was used to form an implant precursorfrom the liquid polymer mixture. The porous polyethylene-substrates oneach side of the case were saturated with about 2.5 mL of sterilesaline. Two polypropylene spacers were placed on the porous polyethylenesubstrate on the lower half of the case (nearest to the trimming grid)such that they were parallel to the hinge of the case and against theedges of the porous polyethylene substrate. A filled container of thepolymer mixture was opened, and the contents (approximately 0.6 g) wereexpelled onto the center of the porous polyethylene substrate betweenthe spacers. The case was closed and latched, and was then reopenedafter six minutes. The semi-rigid article was removed from the porouspolyethylene substrate, placed onto the attached trimming area, andtrimmed to size using a sterile razor blade.

The implant precursor was examined visually; it was opaque, semi-rigid,and flexible. The implant precursor had a two-part structure whichconsisted of a gelatinous, semi-rigid outer layer and a more liquidcenter core. Chemical analysis indicated that the implant precursorcontained about 58% NMP.

EXAMPLE 2 In Vitro Formation of an Implant Precursor

An implant precursor was formed as in Example 1 above, except that thecase remained closed for eight minutes. This article was more rigid thanthe article from Example 1 above.

EXAMPLE 3 In Vitro Formation of an Implant Precursor

An implant precursor was formed as in Example 1 above, except that thecase remained closed for four minutes. This article was less rigid thanthe article from Example 1 above.

EXAMPLE 4 In Vitro Formation of an Implant Precursor with a GlassSubstrate

Two spacers with approximate thicknesses of 430 μm were constructed bygluing two sets of three glass microscope cover slips together. Thesewere placed on a glass microscope slide leaving a space between them.Approximately 0.3 g of the same polymer mixture as in Example 1 was thendispensed onto the microscope slide between the spacers using a syringe.An atomizer was used to spray the polymer mixture with water threetimes. After 30 seconds the water spraying was repeated. After anadditional 30 seconds, another microscope slide was sprayed with waterand then pressed onto the coagulating polymer mass and the spacers. Thissecond microscope slide was held in place for 60 seconds and thenremoved. The coagulating polymer mass was then sprayed with water threetimes and allowed to set for 60 seconds. The three water sprays and 60second set was then repeated. The glass microscope slide and thecoagulating polymer mass were then placed over a grid of 1 mm squares. Asterile razor blade was then used to trim the polymer mass to thedesired size and shape. The cut piece was then sprayed with water threetimes and allowed to set for 60 seconds. The excess water was thenremoved using a gauze pad. The opaque and flexible implant precursor wasthen ready for implantation.

EXAMPLE 5 In Vitro Formation of an Implant Precursor with a GlassSubstrate

A 2 inch×3 inch microscope slide with a 20 mm×20 mm graph inscribed onthe underside is placed on a 2 inch×3 inch×1/4 inch Gray-Lite #14 darkbackground glass. On the top side of the microscope slide were placed 1inch diameter 750 micron stainless steel washers. A washer is placed onthe left and right of the inscribed graph. A polymer mixture prepared asdescribed in Example 1 was then layered over the microscope slide andsmoothed to remove any bubbles or uneven areas. Sterile isotonic salinewas carefully dropped onto the middle of the liquid polymer layer whereit flowed laterally to cover the entire film. The saline was allowed tostay in contact with the polymer mixture for 1 minute at which time theoutside surface or skin became opaque. The excess saline was thencarefully removed by air spray or sponge and the entire process repeatedagain with addition of more polymer mixture and saline to coagulate thepolymer. After the second layer had set for 1 minute, a 1 inch×3 inchregular microscope glass slide moistened with saline solution was placedover the polymer mixture and compressed to the height of the stainlesssteel washers (750 μm). Additional saline was added to the edge of theregular microscope glass slide to saturate the underside of the slide.The compressed material was allowed to set for 10 more minutes. Theregular microscope slide and washers were then removed and the implantprecursor film was cut with a single razor blade to the dimensions ofthe periodontal defect.

EXAMPLE 6 Application of an Implant Precursor to a Periodontal Defect

A mandibular first molar of a 65-year old man was selected for treatmentbecause of long-standing pocket depth and furcation involvement. Duringsurgery, a full-thickness periodontal flap was elevated, the defectscaled and root planed, and the dimensions of the defect measured. Acustomized implant precursor barrier membrane prepared according toExample 5 was applied over the periodontal defect so as to approximatethe level of the crown margin and overlay the osseous margins by 2 to 3mm. The precursor material adhered directly to the tooth and bonewithout the need for suturing in place. The buccal flap was replacedover the defect and the implant precursor and sutured to the lingualtissue. A periodontal dressing material was applied to the surgical areaand systemic antibiotic therapy was used for 7 days. After one week, thefully formed barrier was in place. At one month, the barrier was alsopresent but displaced buccally from the tooth surface because of theformation of granulation tissue between the barrier and the rootsurface. At the 6-month examination, the barrier was no longer evidentand epithelium had grown over the former area of granulation tissue. Theclinical probing measurements at this time showed that the periodontalpocket depth had decreased from 5 mm to 2 mm and the level of attachmentof tissue to the tooth had increased from 7 mm to 4 mm. The horizontalfurcation depth had also decreased from 5 mm to 3 mm. All clinicalmeasurements indicated good tissue regeneration at the defect site.

EXAMPLE 7 Treatment Using an Implant Precursor in Combination with aSupport Layer

A polymer mixture may be prepared as described in Example 1. A thighbone of an anesthetized male rat may be surgically incised to create adefect. Granules of Surgicel® oxidized cellulose may be applied to thedefect to stop the bleeding and to fill in the defect. A precursorimplant prepared as described in Example 1 may be applied over thesurface of the Surgicel™ support layer. The tissue is then replaced andsutured in place. The implant precursor will further solidify to a solidbarrier matrix.

EXAMPLE 8 Treatment in Which the Implant Precursor is Formed In Vivoover a Support Layer

A thigh bone of an anesthetized male rat may be surgically incised tocreate a defect and granules of Surgicel™ oxidized cellulose may beapplied to the defect to stop the bleeding and to fill in the defect. Apolymer mixture prepared according to Example 1 may then be applieddirectly over the surface of the Surgicel™ support layer. The moisturefrom the tissue defect will cause the liquid polymer to partiallysolidify to form the same type of implant precursor as described inExample 1. The soft tissue is then replaced and sutured into place. Theimplant precursor thus formed will further solidify to a solid barriermatrix.

EXAMPLE 9 Treatment with an Implant Precursor Comprising a BiologicalAgent

A polymer mixture may be prepared as described in Example 1. To thismixture may be added 5% by weight doxycycline hyclate. An implantarticle may then be prepared in vivo from the drug/polymer mixture asdescribed in Example 1. The implant article may be placed into aperiodontal defect as described in Example 6. The doxycycline will bedispensed from the solid barrier implant as it degrades and provideprotection against bacterial infection.

EXAMPLE 10 In Vivo Formation of an Implant Precursor in a Bone

A polymer mixture may be prepared as described above in Example 1.

A thigh bone of an anesthetized male rat may be surgically incised, andthe surface of the incision of the bone tissue may be coated with a thinlayer of a phosphate buffered saline (PBS) solution. The polymer mixture(about 1-3 ml) may be dispensed from a syringe or eye dropper onto thesurface of the water-coated bone tissue. The buffer solution (about 1-3ml) may be dispensed onto the layer of the polymer mixture. After 2-5minutes, the polymer mixture will coagulate to form a gelatinous outerlayer with a liquid content of an implant precursor.

The implant precursor may be covered with tissue, and the tissue suturedin place. The implant precursor will gradually solidify to a solidmatrix. After 5-10 days, the implant site may be reopened, and theimplant article mass should have been displaced by the ingrowth of bonetissue.

What is claimed is:
 1. A method of treating a bone tissue defect in ananimal, the method comprising:dispensing a polymer solution onto asupport substrate surrounded by an aqueous medium or body fluid; whereinthe polymer solution comprises a pharmaceutically acceptablebiodegradable, thermoplastic polymer that is insoluble in the aqueousmedium or body fluid; in combination with a biocompatible organicsolvent that solubilizes the polymer and is miscible to dispersible inthe aqueous medium or body fluid; and a bone growth promoting substance,wherein the polymer solution is in a liquid form, wherein the supportsubstrate is a tissue in the animal and the organic solvent diffusesinto the aqueous medium or body fluid and the thermoplastic polymercoagulates to form a solid microporous implant in which the bone growthpromoting substance is incorporated when the polymer solution isdispensed on the support substrate.
 2. The method of claim 1 wherein thebone growth promoting substance is hydroxyapatite.
 3. The method ofclaim 1 wherein the bone growth promoting substance is bone morphogenicprotein.
 4. The method of claim 1, wherein the thermoplastic polymer isselected from the group consisting of polylactides, polyglycolides,polycaprolactones, polyanhydrides, polyamides, polyurethanes,polyesteramides, polyorthoesters, polydioxanones, polyacetals,polyketals, polycarbonates, polyorthocarbonates, polyphosphazenes,polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates,polyalkylene succinates, polymalic acid, polyamino acids, polymethylvinyl ether, chitin, and chitosan.
 5. The method of claim 1, wherein thethermoplastic polymer is selected from the group consisting ofpolylactide, polycaprolactone, and polyglycolide.
 6. The method of claim1, wherein the organic solvent is selected from the group consisting ofN-methyl-2-pyrrolidone, 2-pyrrolidone, ethanol, propylene glycol,propylene carbonate, acetone, acetic acid, ethyl acetate, ethyl lactate,methyl acetate, methyl ethyl ketone, dimethylforamide, dimethylsulfoxide, dimethyl sulfone, tetrahydrofuran, caprolactam,decylmethylsulfoxide, oleic acid, N,N-diethyl-m-toluamide, and1-dodecylazacycloheptan-2-one, and combinations thereof.
 7. Apharmaceutical composition suitable for treating a bone tissue defect inan animal, the composition comprising:a pharmaceutically acceptable,biodegradable, thermoplastic polymer that is insoluble in an aqueousmedium or body fluid; a biocompatible organic solvent that solubilizesthe polymer and is miscible to dispersible in the aqueous medium or bodyfluid; and an effective amount of bone growth promoting substance,wherein the bone growth promoting substance is hydroxyapatite; wherein,the composition is flowable when the composition is placed in contactwith the aqueous medium or body fluid, and upon contacting the bodyfluid or aqueous medium the organic solvent diffuses into the aqueousmedium or body fluid and the thermoplastic polymer coagulates to form asolid microporous implant in situ in which the bone growth promotingsubstance is incorporated.
 8. A pharmaceutical composition suitable fortreating a bone tissue defect in an animal, the composition comprising:apharmaceutically acceptable, biodegradable, thermoplastic polymer thatis insoluble in an aqueous medium or body fluid; a biocompatible organicsolvent that solubilizes the polymer and is miscible to dispersible inthe aqueous medium or body fluid, wherein the solvent is selected fromthe group consisting of N-methyl-2-pyrrolidone, 2-pyrrolidone, dimethylsulfoxide, ethyl lactate and propylene carbonate; and an effectiveamount of bone growth promoting substance; wherein, the composition isflowable when the composition is placed in contact with the aqueousmedium or body fluid, and upon contacting the body fluid or aqueousmedium the organic solvent diffuses into the aqueous medium or bodyfluid and the thermoplastic polymer coagulates to form a solidmicroporous implant in situ in which the bone growth promoting substanceis incorporated.
 9. The composition of claim 8, wherein the bone growthpromoting substance is hydroxyapatite.
 10. The composition of claim 8,wherein the bone growth promoting substance is bone morphogenic protein.11. The composition of claim 8, wherein the thermoplastic polymer isselected from the group consisting of polylactides, polyglycolides,polycaprolactones, polyanhydrides, polyamides, polyurethanes,polyesteramides, polyorthoesters, polydioxanones, polyacetals,polyketals, polycarbonates, polyorthocarbonates, polyphosphazenes,polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates,polyalkylene succinates, polymalic acid, polyamino acids, polymethylvinyl ether, chitin, and chitosan.
 12. The composition of claim 8,wherein the thermoplastic polymer is selected from the group consistingof polylactide, polycaprolactone, and polyglycolide.